Development and maintenance of neurotransmitter phenotype involves a complex interaction of environmental factors guiding the proper expression of the genes for neurotransmitter biosynthetic enzymes. We have recently cloned the cDNA coding for the acetylcholine synthesizing enzyme choline acetyltransferase (ChAT). We now propose a program to study the factors responsible for the proper expression of this enzyme during development. Our approach will be to investigate the levels and cellular location of ChAT mRNA production under normal conditions as well as under a variety of experimentally manipulated developmental conditions. We will also undertake a major effort to identify the important features of this gene for enzymatic activity as well as other regulatory functions. In vitro mutagenesis will be employed to construct altered ChAT DNA. RNA derived from the mutagenized DNA will then be tested for the ability to direct the synthesis of active enzyme in Xenopus oocytes. Mutagenized DNA will also be reintroduced into Drosophila by P-element transformations in order to study its expression in the context of "whole organism" development. The results of our studies should provide a better understanding of the regulatory features of proper neurotransmitter expression. It is hoped that this information will also help in understanding the underlying disease mechanisms which may be involved in the neurotransmitter specific neuro-degenerative disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.